1. Field of the Invention
The invention relates to the areas of microbial genetics and recombinant DNA technology. The invention provides DNA sequences, vectors, microorganisms, and methods useful for inducing and regulating the expression of genes, including those that are involved in amino acid biosynthesis, in bacterial cells.
2. Background Art
Coryneform bacteria are Gram-positive bacteria frequently used for industrial-scale production of amino acids, purines, and proteins. Although coryneform bacteria, particular Corynebacterium species, have been widely used for industrial purposes for many years, the techniques of molecular biology have only recently been employed to augment the usefulness of these organisms in the production of amino acids and other products.
One way to improve the productivity of a microbial strain is to increase the expression of genes that control the production of a metabolite. Increasing expression of a gene can increase the activity of an enzyme that is encoded by that gene. Increasing enzyme activity can increase the rate of synthesis of the metabolic products made by the pathway to which that enzyme belongs. In some instances, increasing the rate of production of a metabolite can unbalance other cellular processes and inhibit growth of a microbial culture. The modified culture will make more product per cell, but will not be able to generate enough cells per volume to show an improvement over the parent strain in a fermentor.
Transcription is the process by which an RNA molecule is synthesized from a DNA template and occurs by the interaction of a multisubunit enzyme complex, known as RNA polymerase, with a DNA molecule. The RNA that is synthesized by this process ultimately directs the production of protein products within the cell. In general, the rate at which RNA is synthesized from DNA, i. e., the transcription rate, directly influences the level of synthesis of the corresponding protein product.
Promoters are DNA sequence elements that regulate the rate at which genes are transcribed. Promoters can influence transcription in a variety of ways. For example, some promoters direct the transcription of their associated genes at a constant rate regardless of the internal external cellular conditions. Such promoters are known as constitutive promoters. In many cases, however, a promoter will direct transcription of its associated gene only under very specific cellular conditions. For example, promoters that turn off gene expression during the growth phase of a microbial culture, but turn on gene expression after optimal growth has been achieved can be used to regulate genes that control production of a metabolite. The new strain will have the same growth pattern as the parent but produce more product per cell. This kind of modification can also improve titer (g product/liter) and yield (g product/g glucose). Nucleotide sequences have been identified that can be used to increase or decrease gene expression in Corynebacterium species. These regulatable promoters can increase or decrease the rate at which a gene is transcribed depending on the internal and/or the external cellular conditions. Frequently, the presence of a factor, known as an inducer, can stimulate the rate of transcription from a promoter. Inducers can interact directly with molecules that, themselves, physically interact with the promoter or with DNA sequences in the vicinity of the promoter. Alternatively, the action of an inducer in stimulating transcription from a promoter may be indirect. Whereas inducers function to amplify the level of transcription from a promoter, there is a class of factors, known as suppressors, that reduce or inhibit transcription from a promoter. Like inducers, suppressors can exert their effects either directly or indirectly.
Besides regulation through inducers and suppressors, certain promoters are regulated by temperature. For instance, a the level of transcription from a promoter may be increased when cells harboring that promoter are grown at a temperature that is greater than the optimum or normal growth temperature for that cell type. Similarly, there are promoters that will enhance gene expression in cells grown at temperatures below the normal growth temperature.
Promoters are found naturally in wild-type cells where they regulate the expression of specific genes. Promoters, however, are also useful as tools of molecular biology in that they can be isolated from their normal cellular contexts and engineered to regulate the expression of virtually any gene.
The use of regulatory sequences from Escherichia coli to control the expression of reporter genes in Corynebacterium have been documented. The lacIq repressor and the tac promoter/reporter genes from E. coli were on plasmids that replicate in Corynebacterium. See, e.g., Morinaga, Y. et al., J. Biotechnol. 5:305–312 (1987). In addition, Ben-Samoun et al., FEMS Microbiology Letters 174:125 (1999), which is incorporated herein by reference in its entirety, disclose the use of the E. coli araBAD promoter and the araC activator on a plasmid which replicates in Corynebacterium glutamicum cells to stimulate the expression of the GFPuv reporter gene only when L-arabinose is present in the growth medium. The authors acknowledge, however, that the level of expression from the araBAD promoter in C. glutamicum is 6.5 fold lower than that which was observed in E. coli, the native species for the promoter.
U.S. Pat. Nos. 5,693,781 and 5,762,299, each of which are incorporated herein by reference in their entireties, disclose the isolation of promoter sequences from the coryneform bacteria, Brevibacterium flavum. Sequences described in these patents were isolated on the basis of their ability to direct expression of a reporter gene in B. flavum at a level that was greater than the expression level observed in B. flavum with the synthetic tac promoter. Also disclosed in U.S. Pat. Nos. 5,693,781 and 5,762,299, each of which are incorporated herein by reference in their entireties, are B. flavum promoters capable of expressing a reporter gene in B. flavum cells when grown in medium containing: (a) ethanol but not glucose, and vice versa; (b) glucose but not fructose; and (c) glucose but not casein hydrolysates/yeast extract/glucose, and vice versa. The novel promoter sequences disclosed in the present application are different from those described in U.S. Pat. Nos. 5,693,781 and 5,762,299.
A limited number of C. glutamicum promoters have been described to date. For example, the C. glutamicum aceA promoter is disclosed in Wendisch et al., Arch Microbiol. 168:262 (1997) and in U.S. Pat. No. 5,700,661 (where it is termed the isocitrate lyase promoter), each of which are incorporated herein by reference in their entireties. In both of these references, the aceA promoter was linked to a reporter gene in transformed C. glutamicum cells, and produced an extracellular protein, not a product of metabolic engineering. Expression of the reporter gene was found to be greater in C. glutamicum transformants that were grown in the presence of acetate than it was for transformants grown in the presence of glucose (Wendisch et al., Arch Microbiol. 168:262 (1997)) or sucrose (U.S. Pat. No. 5,700,661).
Similarly, the aceB promoter from C. glutamicum is disclosed in Wendisch et al., Arch Microbiol. 168:262 (1997) and in U.S. Pat. No. 5,965,391, which is incorporated herein by reference in its entirety. Both of these references describe transcriptional fusions consisting of the aceB promoter region linked to a reporter gene in C. glutamicum transformed cells. Expression of the reporter gene was found to be greater in C. glutamicum transformants grown in acetate-containing medium than it was for transformants grown in glucose-containing medium (Wendisch et al., Arch Microbiol. 168:262 (1997)) or other carbon sources (U.S. Pat. No. 5,965,391).
Reinscheid et al., Microbiology 145:503 (1999), which is incorporated herein by reference in its entirey, discloses a transcriptional fusion between the C. glutamicum pta-ack promoter and a reporter gene (chloramphenicol acetyltransferase). C. glutamicum cells harboring the transcriptional fusion demonstrated enhanced reporter gene expression when grown in acetate-containing medium as compared to transformed cells that were grown in glucose-containing medium.
In Pátek et al., Microbiology 142:1297 (1996), which is incorporated herein by reference in its entirety, several DNA sequences from C. glutamicum, identified on the basis of their ability to promote the expression of a chloramphenicol resistance reporter gene in C. glutamicum cells, are disclosed and compared to one another in an attempt to define a consensus sequence for C. glutamicum promoters.
There is clearly a need for a broader assortment of well-defined Corynebacterium species promoters than has been heretofore described. Such promoters would be useful in the constitutive and/or regulated expression of genes in coryneform cells. For example, a collection of C. glutamicum promoters, regulated by inexpensive carbon sources, would facilitate the industrial-scale production of amino acids and purines in C. glutamicum cells by enhancing the expression of genes that encode components of the biosynthetic pathways for the desired amino acids or purines. Likewise, a versatile array of coryneform promoters would be useful for the industrial scale production of heterologous polypeptides in C. glutamicum cells by stimulating the enhanced expression of genes encoding such heterologous polypeptides.